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Method of making bioethanol by using glucose isomerase mutants

a technology of glucose isomerase and mutants, applied in biofuels, organic chemistry, enzymology, etc., can solve problems that are not applicable in industrial applications, and achieve the effect of improving catalytic activity

Active Publication Date: 2011-09-06
BIORIGHT WORLDWIDE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Nevertheless, the thermostabilities of the thermostable glucose isomerases from these and other bio-resources are still much to be desired as the activities thereof are low, and thus are not applicable to industrial applications.

Method used

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  • Method of making bioethanol by using glucose isomerase mutants

Examples

Experimental program
Comparison scheme
Effect test

example 1

Amplification of Wild-Type Glucose Isomerase and Construction of pGEMT-TS

[0021]Primers T1 and T2 (Table 1) were designed based on the sequence of GenBank L09699 and used to amplify the wild-type glucose isomerase gene from T. saccharolyticum ATCC 49915 (ATCC, USA).

[0022]The amplification condition was: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer T1, 400 nM primer T2, 1.5 U Taq DNA polymerase (Promega, USA), a loopful of T. saccharolyticum colony, and the total volume was adjusted to 50 μl with sterile distilled water.

[0023]The PCR amplification program for the reaction was: 95° C., 3 min; then 40 cycles of 95° C., 50 sec, 50° C., 30 sec, 72° C., 1 min; and finally 72° C., 10 min. The amplified PCR product, about 1.5 kb in length, was ligated into vector pGEMT-Easy to generate pGEMT-TS. The pGEMT-TS was sequenced to determine the DNA sequence of the wild-type glucose isomerase as Seq...

example 2

Site-Directed Mutagenesis of Trp139 of Wild-Type Glucose Isomerase

[0024]The site directed mutagenesis was done as described by Ho et al., Gene 77:51-59, 1989 and White et al., PCR protocol: current methods and applications. Totowa, N.J.: Humana Press 1993.

[0025]With pGEMT-TS (Example 1) as template, the Trp (W) at position 139 of the wild-type glucose isomerase was mutated to Phe (F) to generate glucose isomerase mutant MGI-W139F by PCR amplification using primers 139FF and 139FR (Table 1) and universal primers T1 and T2 (Example 1).

[0026]Fragment T1FR was amplified using primer pair T1 and 139FR. Fragment FFT2 was amplified using primer pair 139FF and T2. The amplification condition was: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer T1 and 400 nM primer 139FR (for fragment T1FR) or 400 nM primer T2 and 400 nM primer 139FF (for fragment FFT2), 1.5 U Pfu DNA polymerase (Promega, USA), ...

example 3

Site-Directed Mutagenesis of Arg182 of Glucose Isomerase

[0027]The site directed mutagenesis was done as described by Ho et al., Gene 77:51-59, 1989 and White et al., PCR protocol: current methods and applications. Totowa, N.J.: Humana Press 1993.

[0028]Using pGEMT-TS (Example 1) as template, the Arg (R) at position 182 of the wild-type glucose isomerase was mutated to Ala (A) to generate glucose isomerase mutant MGI-R182A by PCR amplification with site-directed primers 182AF and 182AR (Table 1) and universal primers T1 and T2 (Example 1).

[0029]Fragment T1AR was amplified using primer pair T1 and 182AR. Fragment AFT2 was amplified using primer pair 182AF and T2. The amplification condition was: 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, 50 μM dATP, 50 μM dTTP, 50 μM dCTP, 50 μM dGTP, 400 nM primer T1 and 400 nM primer 182AR or 400 nM primer T2 and 400 nM primer 182AF, 1.5 U Pfu DNA polymerase, 20 ng pGEMT-TS, and the total volume was adjusted t...

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Abstract

This invention provides use of a series of recombinant Thermoanaerobacterium saccharolyticum glucose isomerases with improved catalytic activity obtained by using recombinant techniques. These mutants comprise at least one amino acid variation at position 87, position 139, position 182, position 187, position 217, position 260, position 276, or position 299, and can be used in the conversion of hemicellulose to ethanol.

Description

FIELD OF THE INVENTION[0001]The present invention relates to molecular biology, and specifically relates to recombinant glucose isomerases with improved activity or both of improved activity and thermostability, the method of preparing the same using recombinant techniques, and use of the same.BACKGROUND OF THE INVENTION[0002]Glucose isomerase (E.C.5.3.1.5 or xylose isomerase) is a key enzyme in the pentose phosphate pathway. It is one of the most important industrial enzymes (Kaneko et al., Bioscience, Biotechnology, and Biochemistry 2000, 64:940-947). In bio-energy industry, people are trying to use the enzyme, together with other key enzymes in the degradation pathway of cellulose and hemicelluloses, to produce bioethanol commercially. For example, the pathway of the enzymatic degradation of xylan to xylulose-5-phosphate is as follows: xylan is converted into xylo-oligosaccharides in the presence of β-1,4-xylanase, xylo-oligosaccharides is further turned into xylose by β-xylosida...

Claims

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Application Information

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Patent Type & Authority Patents(United States)
IPC IPC(8): C12P7/06C12N15/00C12N1/20C12N9/90C07H21/04C12N9/00
CPCC12N9/92C12P7/10C12P19/24Y02E50/16Y02E50/17Y02E50/10
Inventor WANG, JUNFU, RONGZHAOCHEUNG, TIN CHO
Owner BIORIGHT WORLDWIDE
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